ABSTRACT Atrial natriuretic peptide (ANP) is a cardiac hormone that regulates sodium excretion, fluid volume, and vasorelaxation, important factors in the control of blood pressure and blood volume. Interaction of ANP with its receptor guanylyl cyclase-A/natriuretic peptide receptor-A (GC-A/NPRA) produces the intracellular second messenger cGMP, which plays a central role in the pathophysiology of hypertension and cardiovascular disorders. Gaining insight into the intricacies of ANP/NPRA/cGMP signaling pathways is of pivotal importance for understanding both receptor biology and the disease state arising from the abnormal hormone-receptor interplay. The long-term objective of this proposal is directed at elucidating at the molecular level the nature and mode of functioning of NPRA. Recombinant NPRA expressed in transfected cells lacking the endogenous receptor provides a useful system to accomplish this goal. The insights gained from domain mapping of cDNA clone and biochemical analysis of the receptor will be used to elucidate those receptor areas that can be analyzed further by deletion and site-directed mutagenesis in transfected cells to learn what structural components are involved in the functioning of NPRA. This proposal has four major intimately linked goals, all involving a primary focus on NPRA as an approach to gain increased understanding of the normal and abnormal control of cellular and physiological processes. Accordingly, the specific aims entailed within this application are as follows: 1) delineate the molecular determinants in the GC catalytic active-site of NPRA by site-directed mutagenesis and determine the role of ANP/NPRA signaling and physiological function(s) in vascular smooth muscle and mesangial cells transfected with wild-type and carboxyl-terminal mutant receptors, 2) delineate the cellular and molecular regulatory mechanisms of the protein kinase-like domain of NPRA and examine the effect of ANP/ATP on the phosphorylation state and signal transduction activities of the receptor, 3) examine the molecular determinants mediating the functional aspects of NPRA including the dynamics of internalization, trafficking, sequestration, and recycling by fluorescence microscopy in living cells in real time, and 4) study the molecular determinants mediating post-binding events and metabolic turnover including desensitization and down-regulation of NPRA. The proposed studies will provide a comprehensive assessment of the mode of functioning of NPRA at the molecular level, in which the detailed information about the receptor structure and signaling will provide the basis for understanding receptor function and regulation. Ultimately, this knowledge should yield new therapeutic targets and novel loci for the control and treatment of hypertension and cardiovascular disorders.